RESUMO
The microtubule-associated protein MAP1B plays a key role in axon regeneration. We investigated the role of GSK3-mediated MAP1B phosphorylation in local fine-tuning of neurite branching and the underlying microtubule (MT) dynamics. In wildtype adult dorsal root ganglia (DRG) neurons, MAP1B phosphorylation is locally reduced at branching points, and branching dynamics from growth cones and distal neurite shafts is increased upon GSK3 inhibition. While map1b-/- neurites, that display increased branching, are not affected by GSK3 inhibition, transfection of map1b-/- neurons with full-length map1b-cDNA restores the wildtype branching phenotype, demonstrating that MAP1B is a key effector downstream of GSK3. Experiments in mutant mice lacking tyrosinated MTs indicate a preferential association of phospho-MAP1B with tyrosinated MTs. Interestingly, inhibition of GSK3-mediated MAP1B phosphorylation in map1b-cDNA-transfected fibroblasts protects both tyrosinated and acetylated MTs from nocodazole-induced depolymerization, while detyrosinated MTs are less abundant in the presence of MAP1B. Our data thus provide new insight into the molecular link between GSK3, MAP1B, neurite branching and MT stability regulation. We suggest that, at branching points, MAP1B undergoes a fine regulation of both its phosphorylation and sub-cellular amounts, in order to modulate the local balance between acetylated, detyrosinated, and tyrosinated microtubule pools.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neurogênese , FosforilaçãoRESUMO
The profound morphofunctional changes that Schwann cells (SCs) undergo during their migration and elongation on axons, as well as during axon sorting, ensheathment, and myelination, require their close interaction with the surrounding laminin-rich basal lamina. In contrast to myelinating central nervous system glia, SCs strongly and constitutively express the giant scaffolding protein AHNAK1, localized essentially underneath the outer, abaxonal plasma membrane. Using electron microscopy, we show here that in the sciatic nerve of ahnak1(-) (/) (-) mice the ultrastructure of myelinated, and unmyelinated (Remak) fibers is affected. The major SC laminin receptor ß-dystroglycan co-immunoprecipitates with AHNAK1 shows reduced expression in ahnak1(-) (/) (-) SCs, and is no longer detectable in Cajal bands on myelinated fibers in ahnak1(-) (/) (-) sciatic nerve. Reduced migration velocity in a scratch wound assay of purified ahnak1(-) (/) (-) primary SCs cultured on a laminin substrate indicated a function of AHNAK1 in SC motility. This was corroborated by atomic force microscopy measurements, which revealed a greater mechanical rigidity of shaft and leading tip of ahnak1(-) (/) (-) SC processes. Internodal lengths of large fibers are decreased in ahnak1(-) (/) (-) sciatic nerve, and longitudinal extension of myelin segments is even more strongly reduced after acute knockdown of AHNAK1 in SCs of developing sciatic nerve. Together, our results suggest that by interfering in the cross-talk between the transmembrane form of the laminin receptor dystroglycan and F-actin, AHNAK1 influences the cytoskeleton organization of SCs, and thus plays a role in the regulation of their morphology and motility and lastly, the myelination process.
Assuntos
Movimento Celular/fisiologia , Distroglicanas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células de Schwann/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Axônios/diagnóstico por imagem , Axônios/fisiologia , Células Cultivadas , Elasticidade , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Camundongos Knockout , Microscopia de Força Atômica , Bainha de Mielina/fisiologia , Bainha de Mielina/ultraestrutura , Proteínas de Neoplasias/genética , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , RNA Interferente Pequeno/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/fisiopatologia , Nervo Isquiático/ultraestrutura , UltrassonografiaRESUMO
We have previously shown that implantation of a fragmented chitosan hydrogel suspension (chitosan-FPHS) into a traumatic spinal cord lesion in adult rats led to significant axon regrowth and functional recovery, which was associated to a modulation of inflammation. Using an in vitro culture system, we show here that polarization of bone marrow-derived macrophages is indeed modified by direct contact with chitosan-FPHS. Reducing the degree of acetylation (DA) and raising the concentration of chitosan (Cp, from 1.5% to 3%), favors macrophage polarization toward anti-inflammatory subtypes. These latter also migrate and adhere efficiently on low, but not high DA chitosan-FPHS, both in vitro and in vivo, while inflammatory macrophages rarely invade a chitosan-FPHS implant in vivo, no matter the DA. Our in vitro model setup should prove a valuable tool for screening diverse biomaterial formulations and combinations thereof for their inflammatory potential prior to implantation in vivo.
Assuntos
Quitosana , Animais , Materiais Biocompatíveis , Quitosana/farmacologia , Hidrogéis/farmacologia , Ativação de Macrófagos , Macrófagos , RatosRESUMO
Although the prefrontal cortex and basal ganglia are functionally interconnected by parallel loops, cellular substrates underlying their interaction remain poorly understood. One novel approach for addressing this issue is microfluidics, a methodology which recapitulates several intrinsic and synaptic properties of cortico-subcortical networks. We developed a microfluidic device where cortical neurons projected onto striatal neurons in a separate compartment. We exploited real-time (low-resolution/high-output) calcium imaging to register network dynamics and characterize the response to glutamatergic and dopaminergic agents. Reconstructed cortico-striatal networks revealed the progressive appearance of cortical VGLUT1 clusters on striatal dendrites, correlating with the emergence of spontaneous and synchronous glutamatergic responses of striatal neurons to concurrent cortical stimulation. Striatal exposure to the NMDA receptor GluN2A subunit antagonist TCN201 did not affect network rhythm, whereas the GluN2B subunit antagonist RO256981 significantly decreased striatal activity. Dopamine application or the D2/D3 receptor agonist, quinpirole, decreased cortico-striatal synchrony whereas the D1 receptor agonist, SKF38393, was ineffective. These data show that cortico-striatal networks reconstructed in a microfluidic environment are synchronized and present characteristics close to those of their in situ counterparts. They should prove instructive for deciphering the molecular substrates of CNS disorders and evaluating the actions of novel therapeutic agents.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Imagem Molecular , Vias Neurais , Animais , Sinalização do Cálcio , Progressão da Doença , Técnicas In Vitro , Camundongos , Microfluídica/métodos , Imagem Molecular/métodos , Neurônios/fisiologia , Receptores de Dopamina D2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Recovery from traumatic spinal cord injury (SCI) usually fails due to a cascade of cellular and molecular events that compromise neural tissue reconstitution by giving rise to glial scarring and cavity formation. We designed a scaffold material for SCI treatment containing only chitosan and water as fragmented physical hydrogel suspension (Chitosan-FPHS), with defined degree of acetylation (DA), polymer concentration, and mean fragment size. Implantation of Chitosan-FPHS alone into rat spinal cord immediately after a bilateral dorsal hemisection promoted reconstitution of spinal tissue and vasculature, and diminished fibrous glial scarring: with astrocyte processes primarily oriented towards the lesion, the border between lesion site and intact tissue became permissive for regrowth of numerous axons into, and for some even beyond the lesion site. Growing axons were myelinated or ensheathed by endogenous Schwann cells that migrated into the lesion site and whose survival was prolonged. Interestingly, Chitosan-FPHS also modulated the inflammatory response, and we suggest that this might contribute to tissue repair. Finally, this structural remodeling was associated with significant, long-lasting gain in locomotor function recovery. Because it effectively induces neural tissue repair, Chitosan-FPHS biomaterial may be a promising new approach to treat SCI, and a suitable substrate to combine with other strategies.
Assuntos
Axônios/fisiologia , Quitosana/uso terapêutico , Hidrogel de Polietilenoglicol-Dimetacrilato/uso terapêutico , Regeneração Nervosa , Traumatismos da Medula Espinal/terapia , Alicerces Teciduais , Animais , Axônios/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/uso terapêutico , Quitosana/farmacologia , Cicatriz/terapia , Feminino , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Locomoção , Bainha de Mielina/fisiologia , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Células de Schwann/fisiologia , Água/químicaRESUMO
During development, microtubule-associated protein 1B (MAP1B) is one of the earliest MAPs, preferentially localized in axons and growth cones, and plays a role in axonal outgrowth. Although generally downregulated in the adult, we have shown that MAP1B is constitutively highly expressed in adult dorsal root ganglia (DRGs) and associated with central sprouting and peripheral regeneration of these neurons. Mutant mice with a complete MAP1B null allele that survive until adulthood exhibit a reduced myelin sheath diameter and conductance velocity of peripheral axons and lack of the corpus callosum. Here, to determine the function of MAP1B in axonal regeneration, we used cultures of adult DRG explants and/or dissociated neurons derived from this map1b-/- mouse line. Whereas the overall length of regenerating neurites lacking MAP1B was similar to wild-type controls, our analysis revealed two main defects. First, map1b-/- neurites exhibited significantly (twofold) higher terminal and collateral branching. Second, the turning capacity of growth cones (i.e., "choice" of a proper orientation) was impaired. In addition, lack of MAP1B may affect the post-translational modification of tubulin polymers: quantitative analysis showed a reduced amount of acetylated microtubules within growth cones, whereas the distribution of tyrosinated or detyrosinated microtubules was normal. Both growth cone turning and axonal branch formation are known to involve local regulation of the microtubule network. Our results demonstrate that MAP1B plays a role in these processes during plastic changes in the adult. In particular, the data suggest MAP1B implication in the locally coordinated assembly of cytoskeletal components required for branching and straight directional axon growth.
Assuntos
Axônios/fisiologia , Gânglios Espinais/citologia , Proteínas Associadas aos Microtúbulos/fisiologia , Regeneração Nervosa , Neurônios/fisiologia , Animais , Axônios/ultraestrutura , Células Cultivadas , Gânglios Espinais/ultraestrutura , Cones de Crescimento/fisiologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Bainha de Mielina/patologia , Neuritos/ultraestrutura , Plasticidade Neuronal , Neurônios/ultraestruturaRESUMO
We recently reported that immature porcine Leydig cells express both somatostatin (SRIF) and SRIF receptor type-2 (sst-2) transcripts. The present study was therefore undertaken to assess whether SRIF might exert autocrine actions on these cells through sst2A receptor, one of the two sst2 isoforms known to exert important neuroendocrine and endocrine functions. Using a polyclonal antibody directed towards the C-terminal tail of the sst2A receptor subtype, receptor immunoreactivity was detected in a subpopulation of Leydig cells and spermatogonia. To address the physiological correlates of this expression we then studied the possible involvement of sst2 receptor in the regulation of testosterone secretion. Functional assays showed that the sst2 agonist octreotide inhibited both basal and hCG-stimulated testosterone secretion by testosterone pretreated Leydig cells. To assess whether sst2 receptor expression might be regulated by testosterone, we performed a semi-quantitative RT-PCR analysis of sst2 mRNA expression in Leydig cells cultured in the presence or in the absence of the androgen. A significant increase in sst2 receptor transcripts was observed in testosterone-treated cells. Taken together, these data suggest that SRIF can inhibit testosterone secretion through the sst2A receptor. The mechanism of the local inhibitory actions of SRIF is probably autocrine since immature porcine Leydig cells express SRIF itself and it might involve testosterone-induced increase of sst2 receptor expression in immature Leydig cells.
Assuntos
Células Intersticiais do Testículo/metabolismo , Receptores de Somatostatina/biossíntese , Somatostatina/fisiologia , Suínos/fisiologia , Testosterona/metabolismo , Animais , Comunicação Autócrina , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Gonadotropina Coriônica/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Octreotida/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Somatostatina/genética , Receptores de Somatostatina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/agonistas , Somatostatina/biossíntese , Testículo/citologia , Testículo/crescimento & desenvolvimentoRESUMO
Microtubule-associated protein-1B (MAP1B), and particularly its phosphorylated isoform MAP1B-P, play an important role in axonal outgrowth during development of the mammalian nervous system and have also been shown to be associated with axonal plasticity in the adult. Here, we used antibodies and mRNA probes directed against mammalian MAP1B to extend our analysis to fish species, trout (Oncorhynchus mykiss), at different stages of development. The specificity of the cross-reaction of our anti-total-MAP1B/MAP1B-P antibodies was confirmed by Western blotting. Trout MAP1B-like proteins exhibited about the same apparent molecular weight (320 kDa) as rat-MAP1B. Immunohistochemistry and in situ hybridization analysis performed on hindbrain and spinal cord revealed the presence of MAP1B in neurons and some glial subpopulations. Primary sensory neurons and motoneurons maintain high levels of MAP1B expression from early stages throughout adulthood, as has been shown for mammals. Unlike mammals, however, MAP1B and axon-specific MAP1B-P continue to be strongly expressed by hindbrain neurons projecting into spinal cord, with the important exception of Mauthner cells. MAP1B/MAP1B-P immunostaining were also detected elsewhere within the brain, including axons of the retino-tectal projection. This obvious difference between adult fish and mammals is likely to reflect the capacity of fish for continued growth and regeneration. Our results suggest that MAP1B/MAP1B-P expression is generally maintained in neurons known to regenerate after axotomy. The regenerative potential of the adult nervous system may in fact depend on continued expression of neuron-intrinsic growth related proteins, a feature of MAP1B that appears phylogenetically conserved.
Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Sistema Nervoso Central/crescimento & desenvolvimento , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Proteínas Associadas aos Microtúbulos/genética , Oncorhynchus mykiss , FosforilaçãoRESUMO
Upregulation of extracellular chondroitin sulfate proteoglycans (CSPG) is a primary cause for the failure of axons to regenerate after spinal cord injury (SCI), and the beneficial effect of their degradation by chondroitinase ABC (ChABC) is widely documented. Little is known, however, about the effect of ChABC treatment on astrogliosis and revascularization, two important factors influencing axon regrowth. This was investigated in the present study. Immediately after a spinal cord hemisection at thoracic level 8-9, we injected ChABC intrathecally at the sacral level, repeated three times until 10 days post-injury. Our results show an effective cleavage of CSPG glycosaminoglycan chains and stimulation of axonal remodeling within the injury site, accompanied by an extended period of astrocyte remodeling (up to 4 weeks). Interestingly, ChABC treatment favored an orientation of astrocytic processes directed toward the injury, in close association with axons at the lesion entry zone, suggesting a correlation between axon and astrocyte remodeling. Further, during the first weeks post-injury, ChABC treatment affected the morphology of laminin-positive blood vessel basement membranes and vessel-independent laminin deposits: hypertrophied blood vessels with detached or duplicated basement membrane were more numerous than in lesioned untreated animals. In contrast, at later time points, laminin expression increased and became more directly associated with newly formed blood vessels, the size of which tended to be closer to that found in intact tissue. Our data reinforce the idea that ChABC injection in combination with other synergistic treatments is a promising therapeutic strategy for SCI repair.
Assuntos
Astrócitos/efeitos dos fármacos , Condroitina ABC Liase/farmacologia , Traumatismos da Medula Espinal/patologia , Remodelação Vascular/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Western Blotting , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The molecular mechanisms triggering microglial activation after injury to the central nervous system, involving cell-extracellular matrix interactions and cytokine signaling, are not yet fully understood. Here, we report that resident microglia in spinal cord express low levels of the non-integrin laminin receptor precursor (LRP), also found on certain neurons and glial cells in the peripheral nervous system. 37LRP/p40 and its 67-kDa isoform laminin receptor (LR) were the first high-affinity laminin binding proteins identified. While the role of laminin receptor was later attributed to integrins, LRP/LR gained new interest as receptors for prions, and their interaction with laminin seems important for migration of metastatic cancer cells. Using immunohistochemistry and Western blotting, we demonstrate that traumatic spinal cord injury leads to a strong and rapid increase in LRP levels in relation to activated microglia/macrophages. Associated with laminin re-expression in the lesion epicenter, LRP-positive microglia/macrophages exhibit a rounded, ameboid-like shape characteristic of phagocytic cells, whereas in more distant loci they reveal a hypertrophied cell body and short ramifications. The same morphological difference is observed in vitro for purified microglia cultured with or without laminin. Strong, transient upregulation of LRP by activated spinal cord microglia is also induced by transection of the sciatic nerve that affects the spinal cord circuitry without blood-brain barrier dysruption. LRP expression is maximal by 1 week post-lesion, before becoming restricted to dorsal and ventral horns, sites of major structural reorganization. Our findings strongly suggest the involvement of LRP in lesion-induced activation and migration of microglia.
Assuntos
Microglia/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Neuropatia Ciática/metabolismo , Traumatismos da Medula Espinal/metabolismo , África Ocidental , Fatores Etários , Animais , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Isomerismo , Macrófagos/citologia , Macrófagos/metabolismo , Microglia/citologia , Precursores de Proteínas/química , Ratos , Ratos Wistar , Proteínas Ribossômicas/química , Células de Schwann/citologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Neuropatia Ciática/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Regulação para Cima/fisiologiaRESUMO
Spinal cord injury (SCI) results in loss of sensory and motor function because injured axons do not regenerate and neurons that die are not replaced. Nevertheless, there is evidence for spontaneous reorganization of spared pathways (i.e. sprouting) that could be exploited to improve functional recovery. The extent of morphological remodeling after spinal cord injury is, however, not understood. We have previously shown that a phosphorylated form of microtubule-associated protein-1B, MAP1B-P, is expressed by growing axons, but is detected in intact adult SC in fibers exhibiting a somatotopic distribution of myelinated sensory fibers. We now demonstrate that after adult SCI, MAP1B-P is up-regulated in other classes of axons. We used immunohistochemistry to show changing levels and distributions of MAP1B-P after a right thoracic hemisection of adult rat spinal cord. MAP1B-P labeling suggests rearrangements of the axonal circuitry that go well beyond previous descriptions. MAP1B-P-positive fibers are present in ectopic locations in gray matter in both dorsal and ventral horns and around the central canal. Double staining reveals that primary sensory and descending serotonergic and corticospinal axons are MAP1B-P positive. In white matter, high MAP1B-P expression is found on terminal enlargements near the injury, reflecting retraction of transected axons. MAP1B-P also accumulates in pre-apoptotic neuronal somata axotomized by the lesion, indicating association of MAP1B-P not only with axon extension and retraction, but also with neuronal degeneration. Finally, we provide evidence that MAP1B phosphorylation is correlated with activation of JNK MAP-kinase, providing a step towards unraveling the mechanisms of regulation of this plasticity-related cytoskeletal protein.
Assuntos
Axônios/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Degeneração Neural/patologia , Neurônios/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Axônios/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Imuno-Histoquímica , MAP Quinase Quinase 4/metabolismo , Fibras Nervosas/patologia , Fibras Nervosas/fisiologia , Neurônios/metabolismo , Fosforilação , Ratos , Ratos Wistar , Medula Espinal/citologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
To investigate the molecular basis for the poor regenerative capacity of the mammalian central nervous system (CNS) after injury, we searched for genes whose expression was affected by an adult rat spinal cord hemi-section. Differential screening of a rat spinal cord expression library was performed using polyclonal antibodies raised against lesioned spinal cord tissue. A striking overexpression was found for ahnak, encoding a 700-kDa protein, in normal CNS present only in the blood-brain barrier (BBB) forming vascular endothelial cells. Indeed, very early after spinal cord injury (SCI), high levels of membrane-associated AHNAK are observed on non-neuronal cells invading the lesion site. With time, AHNAK distribution spreads rostrally and caudally concomitant with the process of tissue inflammation and axon degeneration, delineating the interior surface of cystic cavities, mainly in front of barrier-forming astrocytes. Strong overexpression is also observed on vascular endothelial cells reacting to BBB breakdown. Based on our detailed analysis of its spatiotemporal and cellular expression, and its previously described function in BBB, we suggest that AHNAK expression is associated with cell types displaying tissue-protective barrier properties. Our study may thus contribute to the elucidation of the precise molecular and cellular events that eventually render traumatic spinal cord tissue non-permissive for regeneration.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Fisiológica , Regeneração Nervosa , Traumatismos da Medula Espinal , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/citologia , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Biblioteca Gênica , Humanos , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Expression of the cell adhesion molecule TAG-1 is down-regulated in adult brain, with the exception of certain areas exhibiting structural plasticity. Here, we present evidence that TAG-1 expression persists also in adult rat spinal cord and dorsal root ganglia (DRG), and can be up-regulated after injury. On Western blots of adult tissue, TAG-1 is detected as a 135-kDa band, with an additional specific 90-kDa band, not present in developing tissue. TAG-1 expression is found both in DRG neurons and in Schwann cells, particularly those associated with the peripherally projecting DRG processes. Quantitative in situ hybridization revealed that TAG-1 expression is significantly higher in small neurons that give rise to unmyelinated fibers, than in large DRG neurons. The regulation of TAG-1 was then examined in two different lesion paradigms. After a sciatic nerve lesion, TAG-1 expression is not up-regulated in DRG neurons, but decreases with time. At the lesion site, reactive Schwann cells up-regulate TAG-1, as demonstrated by both immunohistochemistry and in situ hybridization. In a second paradigm, we injected kainic acid into the spinal cord that kills neurons but spares glia and axons. TAG-1 is up-regulated in the spinal neuron-depleted area as well as in the corresponding dorsal and ventral roots, associated with both target-deprived afferent fibers and with the non-neuronal cells that invade the lesion site. These results demonstrate a local up-regulation of TAG-1 in the adult that is induced in response to injury, suggesting its involvement in axonal re-modelling, neuron-glia interactions, and glial cell migration.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Degeneração Neural/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Neuropatia Ciática/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Contactina 2 , Proteínas de Ligação a DNA/metabolismo , Proteína 2 de Resposta de Crescimento Precoce , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Indóis/metabolismo , Ácido Caínico , Proteínas Associadas aos Microtúbulos/metabolismo , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas S100/metabolismo , Células de Schwann , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A peripheral nerve lesion induces sprouting of primary afferents from dorsal root ganglion (DRG) neurons into lamina II of the dorsal horn. Modifications of the environment in consequence to the axotomy provide an extrinsic stimulus. A potential neuron-intrinsic factor that may permit axonal sprouting is microtubule-associated protein 1B (MAP1B) in a specific phosphorylated form (MAP1B-P), restricted to growing or regenerating axons. We show here that both in rat and mouse, a sciatic nerve cut is rapidly followed by the appearance of MAP1B-P expression in lamina II, increasing to a maximum between 8 and 15 days, and diminishing after three months. Evidence is provided that sprouting and induction of MAP1B-P expression after peripheral injury are phenomena concerning essentially myelinated axons. This is in accordance with in situ hybridization data showing especially high MAP1B-mRNA levels in large size DRG neurons that give rise to myelinated fibers. We then employed a second lesion model, multiple rhizotomy with one spared root. In this case, unmyelinated CGRP expressing fibers do indeed sprout, but coexpression of MAP1B-P and CGRP is never observed in lamina II. Finally, because a characteristic of myelinated fibers is their high content in neurofilament protein heavy subunit (NF-H), we used NF-H-LacZ transgenic mice to verify that MAP1B-P induction and central sprouting were not affected by perturbing the axonal organization of neurofilaments. We conclude that MAP1B-P is well suited as a rapidly expressed, axon-intrinsic marker associated with plasticity of myelinated fibers.
Assuntos
Proteínas Associadas aos Microtúbulos/biossíntese , Neurônios Aferentes/metabolismo , Rizotomia , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/análise , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios Aferentes/química , Fosforilação , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Rizotomia/métodos , Rizotomia/estatística & dados numéricosRESUMO
The 67-kDa LR protein was originally discovered as a non-integrin laminin receptor. Several more recent in vitro studies demonstrated the function of 67-kDa LR and its related 'precursor' form 37-kDa LRP as receptors of cellular prion protein and their implication in abnormal prion protein propagation in vitro. In addition, expression of both proteins was shown to increase considerably in the brain of scrapie-infected mice and hamsters. While LRP/LR are thus likely to play important roles in neuronal cell adhesion, survival and homeostasis and during pathological disorders, little is known so far about their fine cellular distribution in adult central nervous system. Using immunocytochemistry and western blotting, we show here that the 67-kDa LR is the major receptor form in adult rat brain and spinal cord, expressed within the cytoplasm and at the plasma membrane of most neurons and in a subset of glial cells. The overall distribution of LR correlates well with that reported for laminin-1 but also with brain regions classically associated with prion-related neurodegeneration. In contrast to LR, the 37-kDa LRP form is much less abundant in adult than in postnatal central nervous system. Characterization of a novel antibody allowed us to study the distribution across tissues of cell membrane-associated LRP. Interestingly, this form is almost exclusively found on a subclass of parvalbumin-immunoreactive cortical interneurons known to degenerate during the early stages of Creutzfeldt-Jakob disease. Our demonstration of local differences in the expression of particular LRP/LR isoforms may be a first step towards unraveling their specific molecular interactions.